Elucidation of 17β-Estradiol (E2) Role in the Regulation of Corpus Luteum Function in Mammals : Analysis of IGFBP5 Expression during Ea-mediated Actions

Abstract

Corpus luteum is a transient endocrine structure formed from the ruptured ovarian follicle. Its main function is to secrete P4, a pro-gestational hormone, essential for establishment and maintenance of pregnancy in mammals. The modulators of CL structure and function are classified as trophic and lytic factors. The luteotrophic factors include pituitary hormones, growth factors, intra luteal factors and cytokines, while luteolytic factors include PGF2α and oxytocin. The interplay between luteotrophic and luteolytic factors regulates luteal steroidogenesis. The precise timing of expression of various enzymes/proteins required for synthesis and metabolism of P4 constitutes an important process in the overall regulation of CL function. The three hormones LH/CG, E2 and PRL are regarded as luteotrophic factors crucial for control of CL function in mammals. Depending on species, either individually or all three hormones in the form of luteotrophic complex have been shown to participate in the regulation of CL function. In addition to the well-established endocrine role of E2, its secretion is the hallmark of the ovulating follicle, has an important role in the intraovarian growth, differentiation and survival of cells. Chapter I provides a comprehensive review of literature on CL structure and function with emphasis on factors that influence its growth, development, function and demise in bovines and rodents. In Chapter II, studies have been carried out to examine 20α-HSD expression and its activity in the CL of buffalo cow. During induced and spontaneous luteolysis, rapid fall in circulating P4 is one of the early signs of initiation of luteolytic process in several species. In rodents, it is well recognized that during luteolysis, P4 is catabolized into inactive metabolite, 20α-OHP by the reaction of 20α-HSD enzyme during luteolysis. Experiments were carried out to determine 20α-HSD expression and activity throughout the luteal phase and during induced luteolysis in the buffalo cow. Circulating P4 concentration declined rapidly in response to PGF2α treatment, but HPLC analysis of serum samples did not reveal changes in circulating 20α-OHP levels in buffalo cows. In contrast, pseudo pregnant rats receiving PGF2α treatment showed higher 20α-OHP levels at 24 h post treatment. qPCR expression of 20α-HSD in CL during different stages of luteal phase and PGF2α-treated buffalo cows was carried out and higher expression of 20α-HSD was observed at 3 and 18 h post treatment, but its activity was not altered post PGF2α treatment at other time points examined. The expression of the transcription factor Nurr77 which is involved in increased expression of 20α-HSD increased several fold 3 h post PGF2α treatment similar to the observation in PGF2α-treated pseudo pregnant rats. The results suggested that the synthesis rather than catabolism of P4 appears to be primarily affected by PGF2α treatment in buffalo cows in contrast to increased metabolism of P4 as seen in rodents. In bovines, to date no luteotropic actions for E2 has been demonstrated and whether E2 has direct effect on CL function has also not been reported. Expression of CYP19A1 gene that encodes aromatase enzyme although gets down regulated post ovulation but its expression recovers in the CL and also E2 biosynthesis has been reported in the bovine CL. Recently it was observed that CYP19A1 expression was consistently down regulated following administration of luteolytic dose of PGF2α. Experiments were conducted to examine the expression of ERα and ERβ in the CL throughout the buffalo estrous cycle as well as examined the luteal E2 levels post PGF2α treatment. The results indicated that ER expression was detectable during different stages of CL and that circulating and luteal E2 levels declined post PGF2α treatment. It was hypothesized that decrease in luteal E2 levels leads to down regulation of ER signaling and changes in expression of E2 responsive genes in the CL. To test the hypothesis, 89 genes which were regarded as E2 responsive genes were selected and the previously published global gene expression data of the buffalo CL was mined for E2 responsive genes. It was observed that 57 of 89 genes regarded as E2 responsive genes were found to be differentially expressed. Since non pregnant buffalo CL is not regarded as major site of E2 production, to validate the authenticity of differentially E2 expressed genes post PGF2α, CL of another species, the macaque, which is known to secrete abundant E2 was included for the analysis. Incidentally, the global gene expression data for the PGF2 α treated macaques (in which CYP19A1 gene expression also gets down regulated) has previously been reported from the laboratory. Here again, it was observed that nearly 79 of 89 genes were identified to be differentially expressed. To further determine the consequences of decreased ER signaling, molecules associated with survival and apoptosis were examined. The results indicated decreased expression (both mRNA and protein levels) of Akt, Bax and Bcl-2 genes. The results suggested an important role for E2 on CL function in the buffalo cow. In Chapter III, several experiments were conducted in another model system, pregnant rat, in which aromatase expression and therefore E2 production is high in the CL. Experiments were conducted to examine the effects of E2 inhibition and E2 replacement on the expression of genes. For this purpose, pregnant rats were treated with a specific aromatase inhibitor on day 12-15 of pregnancy. Together with AI, exogenous E2 was administered to another group of pregnant rats. The CL collected from different groups of rats on day 16 of pregnancy was subjected to microarray analysis. The analysis post validation of microarray data has shown that clusters of genes could be segregated into various pathways involving luteal steroidogenesis, immune system, various growth factors and apoptotic processes, all directed towards the regulation of CL function. The involvement of E2 in luteal cell proliferation and lipid deposition well corroborated with protein levels for cyclin D1 and ki67 and the results of oil red O staining, respectively. There have been reports implicating PI3K/Akt signaling in cyclin D1 accumulation, but mechanism of action does not appear to involve transcriptional activation of cyclin D1. The results of the present study indicate a decrease in cyclin D1 protein levels due to inhibition of PI3K/Akt signaling by AI treatment which is prevented upon administration of E2 during AI treatment. The findings provide a comprehensive overview for the mechanisms associated with the cell survival, progression, etc. The bioinformatics approach provided complete landscape of functional changes affected by the upstream regulators of genes associated with survival and apoptosis. Also, the findings further strengthen the hypothesis of involvement of E2 in the regulation of CL function by way of activation of Akt, the primary mediator of PI3K signaling in the regulation of cellular component that affect cell survival. In the present study, IGFBP5 which was up regulated during luteal inhibition of E2 with AI treatment was selected for further studies. Although IGFBP5 is known to be associated with follicular atresia in the rat ovary, there is limited data for the involvement of IGFBP5 in either a growth stimulatory or inhibitory action on ovarian cells. Based on present findings, a causal link between reduced ERα transcriptional activities resulting in inhibition of Akt/PKB in the presence of IGFBP5 expression could be proposed. Further, the cellular hypertrophy mediated by E2 has been speculated due to increased proliferation of vascular endothelial cells, blood supply and thus nutrients. E2, together with PRL and placental lactogens, regulates steroidogenesis and cell hypertrophy in the rat CL of pregnancy. In CL, the prominent IGFBP5 mRNA expression in different types of luteal cells has not been reported. The mRNA expression for IGFBP5 across the two types of luteal cells showed higher expression in SLC. Hence, in the present study, it has been speculated that prevention of conversion of SLC to LLC due to lack of E2 biosynthesis in presence of AI might be acting as a source for the increased IGFBP5 levels during mid pregnancy in rat CL and brings about changes associated with lack of E2. Various receptor studies on rat CL have demonstrated the lack of progesterone receptor (PR) mRNA expression in the rat CL negating its involvement as an autocrine/paracrine regulator of CL function through an intracellular receptor, but the involvement of non-PR involvement in mediating such mechanism further strengthens the role of ERs. The luteotrophic complex formation in pregnant rat principally by PRL and E2 has been discussed at length in Chapter III. PRL appears to maintain luteal ER content in the CL during rat pregnancy which further determines the luteotrophic and luteolytic actions of E2. Further, study on expression of E2 responsive genes would help in identifying E2 regulating molecules to get a clear picture on the role of E2 in understanding regulation of the CL function. The interaction of E2 with growth factor signaling including the IGF pathway has been well established in different species and this interaction is tightly linked to ERα expression, an observation interpreted as physiological coupling of growth factor and stress signaling pathways. Attempts were made towards understanding cross talk between the E2 signaling and the IGF1 signaling in few experiments carried out in Chapter IV. Based on the results, it can be proposed that a causal link exists between reduced ERα transcriptional activity and inhibition of Akt/PKB in the presence of IGFBP5. The present study has shown the activity of IGF on ERα activity mediated partly via PI3K/Akt pathway. Hence, the finding further speculates that inhibitory effect of IGFBP5 on E2 induced ERα function was due to sequestration of IGF1, possibly present in serum or produced within the cells. Another striking observation was the down regulation of glucocorticoid receptor (GR) gene, NR3C1, in the data of earlier studies [Priyanka, 2009, GEO accession number GSE8371 and Kunal, 2014, GEO accession number GSE27961] and the present study has been compared and discussed in this thesis. Glucocorticoids provide key signals for differentiation of fetal and placental tissues. Therefore, regulation of glucocorticoid access to the placenta and fetus is recognized as an important determinant of prognosis outcome and subsequent development of the postnatal phenotype. Differential regulation of these genes in CL post E2 deprivation and replacement further emphasize the regulation of CL via various biological, cellular and molecular functions. Interestingly, besides transcriptional regulation of IGF axis components, E2 activated ERα also rapidly influence the activity of IGF axis related to signaling proteins in a non-genomic manner, especially by the PI3K/Akt pathway. PI3K/Akt pathway analysis has been carried out in E2 inhibition and replacement experiments. To further confirm the observations of E2 and growth factor interaction, experiments have been set up with exogenous GH for increasing circulating levels of IGF in the system. The findings suggest that the non-genomic signaling pathway activated by the phosphorylation of ERα induced by E2 gets inhibited in the presence of AI result in increased expression of IGFBP5. The reduction in circulating IGF1 in pregnant rats may be associated with the effect on IGFBP, important for determining biological action of IGF1. The changes observed in the present study emphasize the exclusive effects of the IGFBP5 on the CL function brought about perturbations in luteal E2 content. The experiments described in the present thesis aim at understanding the mechanism responsible for decreased serum and luteal P4 post PGF2α treatment in buffalo cows, i.e. whether PGF2α acts on biosynthetic or catabolic process of P4. In the present study, experiments were designed to elucidate the role of E2 in regulation of CL function, since down regulation of CYP19A1 gene mRNA was one of the early events observed in buffalo cows post PGF2α treatment. This line of research work was extended to rodents, a species that secretes high levels of E2 during pregnancy. Genome wide transcriptional changes data revealed differential expression of several E2 responsive genes following E2 inhibition and replacement treatments. The results revealed importance of ER-mediated PI3K/Akt signaling essential for regulation of many transcriptional regulatory molecules in the CL and an interesting involvement of IGFBP5 as a link between E2 and IGF signaling. These findings further provide an insight into the role of IGFBP5 in E2-mediated actions in rat CL during pregnancy. In conclusion, the present findings suggest inhibitory effect of IGFBP5 on E2-induced ERα function and hence, its selection as a target molecule for regulation of CL function and for many beneficial processes involved in anti-carcinogenic properties can be thought of.